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Investigation of Alkaline Phosphatase Enzyme of a Novel Bacillus Species Isolated from Rhizospheric Soil of Potato Field M

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Investigation of Alkaline Phosphatase Enzyme of a Novel Bacillus Species Isolated from Rhizospheric Soil of Potato Field M Bhattacharjee et al RJLBPCS 2018 www.rjlbpcs.com Life Science Informatics Publications Original Research Article DOI - 10.26479/2018.0402.10 INVESTIGATION OF ALKALINE PHOSPHATASE ENZYME OF A NOVEL BACILLUS SPECIES ISOLATED FROM RHIZOSPHERIC SOIL OF POTATO FIELD M. Bhattacharjee1*, M. Banerjee2, P. Mitra3, A. Ganguly4 1.Deptt.of Biotechnology, Heritage Institute of Technology, Chowbaga Road, Ananadapur, P.O-East Kolkata Township, Kolkata-700107, West Bengal. 2. Deptt.of Biochemistry, Triveni Devi Bhalotia (TDB) College, Raniganj, Dist-West Bardhhaman, West Bengal-713347. 3. Deptt.of Biochemistry, Duragapur College of Commerce and Science (DCCS), G.T.Road, Rajbandh, Durgapur-713212, West Bardhhaman Dist., West Bengal. 4. Deptt.of Microbiology, Bankura Sammilani College, Kenduadihi, Bankura, West Bengal-722102 ABSTRACT: The present study reports isolation and biochemical/physiological characterization of a novel Bacillus strain (designated as PB-1) from the rhizospheric soil of potato field that exhibited phosphate solubilization activity. The organism grows optimally at pH 6.0-9.0; at temperature 28-370C and in presence of 1% NaCl. These data indicate that the bacterium is mostly a neutrophile, mesophile and non-halotolerant in nature that matches with the features of a typical soil-borne bacterium. It was also seen that the organism was able to ferment various types of carbohydrates including both mono-and disaccharides. In the second phase of the study, partial purification and characterization of the extracellular alkaline phosphatase enzyme from the Bacillus PB-1 strain was carried out. It was found that the said enzyme was optimally active at 400C temperature and at pH range 8.0-9.0. The enzyme activity increased consistently with increase in the concentration of inorganic phosphate in the growth medium up to 4%. Concentration of phosphate above that did not affect the enzyme activity any more suggesting that the substrate binding site of the enzyme may become saturated at 4% concentration of phosphate which is an important aspect to understand the kinetics of the enzyme. The effect of various carbon sources on the enzyme activity (production) was also studied and was observed that fructose was the best inducer of the enzyme rather than glucose which is conventionally used in the Pikovskaya’s medium for detecting phosphatase activity. The production of phosphatase enzyme by this rhizospheric strain of Bacillus explores its pivotal role as a PGPR that increases soil fertility by de-mineralization of © 2018 Life Science Informatics Publication All rights reserved Peer review under responsibility of Life Science Informatics Publications 2018 March – April RJLBPCS 4(2) Page No.129 Bhattacharjee et al RJLBPCS 2018 www.rjlbpcs.com Life Science Informatics Publications bound phosphates and help in promoting plant growth. Further experiments on the function of this bacterium on inducing crop growth and its molecular identification by 16srRNA sequencing remain to be done in future as an extension of the current work. KEYWORDS: Rhizosphere, PGPR, Bacillus, Alkaline Phosphatase, Characterization, Purification *Corresponding Author: Mainak Bhattacharjee Deptt.of Biotechnology, Heritage Institute of Technology, Chowbaga Road, Ananadapur, P.O-East Kolkata Township, Kolkata-700107, West Bengal. * Email Address: [email protected] 1.INTRODUCTION The genus Bacillus is widely distributed in nature, even in extreme environmental conditions. This genus is comprised of Gram-positive, rod-shaped, endospore-forming non-capsulated and aerobic bacteria that belong to the family Firmicutes which are phylogenetically and phenotypically heterogenous (Claus and Berkeley, 1986). Rhizosphere soil harbors many different species of bacteria including the Bacillus that might exert a positive effect on the growth of the plant and are, therefore, classified as Plant Growth Promoting Rhizobacteria (PGPR). Among the diverse types of PGPR, Bacillus being a spore-former has advantage over the other non-spore forming bacteria due to their resistant nature to high temperatures and strong chemicals used in the form of fertilizers [1]. Various species of Bacillus are also potent sources of many industrially important enzymes one of which is alkaline phosphatase (ALP or ALPase). The ALP enzyme is found to be present in most organisms starting form bacteria to higher animals the main function of which is to release phosphate from organic molecules when inorganic phosphate is limited in the growth environment[2].Alkaline phosphatase (E.C.3.1.3.1) is basically classified as phosphomonoesterases that exert their monoesterase activity through the formation of a phosphoseryl intermediate[2].The enzymes in this group, however, vary in their sizes, substrate specificities and metal ion requirements as co-factors[2]. The enzyme is widely used commercially in gene cloning methods and in other fields of biotechnology, as therapeutic agents and also has other uses in clinical medicine. As for example- one of its uses in clinical medicine is to label monoclonal antibodies during their production [3]. Additionally, in agriculture, phosphorus is considered to be a major nutrient that limits plant (crop) growth [4]. Phosphorus is presently receiving more attention as a non-renewable source (Shen et al., 2011) because of its role in agricultural production. The phosphorus in soil becomes immobilized and thus unavailable to plants even after its application in the form of phosphate fertilizer (Rodriguez and Fraga, 1999). In contrary, there are several Phosphate Solubilizing Bacteria (PSB) present in the rhizospheric soil as a part of the PGPR flora that can release the immobilized phosphate by the © 2018 Life Science Informatics Publication All rights reserved Peer review under responsibility of Life Science Informatics Publications 2018 March – April RJLBPCS 4(2) Page No.130 Bhattacharjee et al RJLBPCS 2018 www.rjlbpcs.com Life Science Informatics Publications production of extracellular phosphatase enzymes (alkaline or acidic)[4] making phosphates utilizable by the crops and thus inducing better growth of the crops. Therefore, the enzyme has significant functions in various aspects of modern science as reflected by its biggest market volume share of $20 million[3]. This makes it an interesting and expanding arena of research to explore new types of ALPs with novel properties suitable for large scale industrial production and applications. The present study initially aims to isolate a species of Bacillus from the rhizospheric soil collected from potato field following its morphological and physiological characterization. In the second phase of the current work, an attempt had been made to screen the phosphatase activity of the isolated strain by growing the same on Pikovskaya’s Agar medium followed by partial purification and characterization of the extracellular alkaline phosphatase enzyme secreted by this particular strain of Bacillus with reference to the enzyme’s optimum temperature, pH as well as effect of phosphate and carbon sources on its activity in order to find out if the enzyme exhibits any novel properties that could be exploited for economical purposes. Similar work performed by Patel F.R.(2016) has led to the extraction and purification of the ALPase enzyme from a halotolerant facultative alkaliphilic strain of Bacillus flexus that has an opportunity to replace ALPases which are now economically obtained from calf intestine, E.coli or shrimps. In addition to this, the work done by Parhamfar M.et al. (2016) has focused on the extraction and characterization of an extracellular acidic phosphatase from Bacillus species. These works were preceded by Kannaiyram S.et al. (2015) who extracted and purified a thermostable ALP enzyme exhibiting optimum activity at 600C from a novel species of Bacillus isolated form rocky soil. Priya D.et al.(2014) has optimized the production of a mesophilic extracellular alkaline phosphatase from Bacillus megaterium. In another such research, Mahesh M.et al.(2010) also purified and characterized a thermostable ALPase enzyme from a novel strain of Bacillus that equally has a promising scope in industries where phosphatase activity at high temperature is required. More recently, Bhattacharjee M.et al. (2017) has reported the extraction and purification of an ALP enzyme from a strain of E.coli isolated from water sample that showed partial thermostable nature. Thus, the investigation of ALP enzyme isolated from a potato rhizosphere soil- borne species of Bacillus seems to be an interesting and worthy research problem that has been done in this study underscoring its importance as a PSB in rhizospheric soil that could play a role as PGPR. 2. MATERIAL AND METHODS Collection of the sample: Rhizospheric soil samples were collected from two different potato fields within 5km.distance located near Arambagh, Hoogly district, West Bengal, India ( latitude 22° 52' 31.6668'' N and longitude 87° 47' 33.5328'' E). Two samples of soil were collected from each plot within air-tight plastic containers. The samples were processed in the lab facility within 12 hrs.of collection. Isolation of bacterial flora from the soil samples: For the purpose of culture, 1g of each of the © 2018 Life Science Informatics Publication All rights reserved Peer review under responsibility of Life Science Informatics Publications 2018 March – April RJLBPCS 4(2) Page No.131 Bhattacharjee et al RJLBPCS 2018 www.rjlbpcs.com Life Science Informatics Publications four soil
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